Retinitis Pigmentosa (RP) is a genetic disease leading to blindness. The prevalence of typical RP is reported to be approximately 1 in 4000 in the United States and currently there is no treatment available for this disease. The final common pathway in RP is typically death of the rod photoreceptors. Several mutations have been found to cause RP, most of them inducing a defect in the retinal pigment epithelium (RPE) or a breakdown of the photoreceptors outer segment disc membranes. The RPE phagocytizes photoreceptor outer segment (OS) membranes that are shed as part of the normal ongoing process of photoreceptor OS renewal. This interaction between the RPE and the photoreceptor OS is crucial for the function and survival of the photoreceptors. Mutations in C-mer proto-oncogene tyrosine kinase, also known as MERTK, have been associated with disruption of the RPE phagocytosis pathway and autosomal recessive RP in humans. The molecular mechanisms of RPE phagocytosis are still unclear. Studies of the internalization of exogenous OS by cultured primary RPE cells suggested a MERTK receptor-specific process since general phagocytosis is not affected, as shown by continued competence of MERTK mutant cells in ingestion of polystyrene beads. However it is clear that the MERTK protein is directly or indirectly involved in the ingestion step of OS phagocytosis since primary dystrophic RPE cells isolated from RCS rat display a dramatic reduction of OS phagocytosis. Here, we propose the development of a new assay to find small molecules that rescue phagocytic function in a cellular model of MERTK loss-of-function in RPE cells. OS preps from bovine retina were labeled with a fluorescent dye and could be efficiently phagocytosed by ARPE19 cells, an RPE cell line. Reduction in the levels of MERTK by RNA interference resulted in a decrease in phagocytic activity providing a cellular phenocopy of a core cell biological event in RP retinal degeneration. This assay has been adapted to 384 well format and successfully quantitated using high content imaging and data processing. A collection of purified secreted proteins and a small compound library have been screened using this method and identified a novel regulators of RPE OS phagocytosis. The proposed work seeks to establish the assay in the more dense 1536 well format to permit screening of several hundred thousand compounds. Compounds identified by this screening method can serve as chemical probes for positive regulation of phagocytosis as well as provide a starting point for the development of novel neuroprotective drugs. PUBLIC HEALTH RELEVANCE: Retinitis Pigmentosa is an incurable genetic disease leading to blindness that strikes approximately 1 in 4000 people in the United States. Patients may be helped by the discovery of new drugs that rescue the function of damaged retinal cells. This proposal seeks to develop new assay technology that could test several hundred thousand compounds to speed the development of new treatments for this devastating disease.